1. Field of the Invention
The present invention relates to a polyhydroxyalkanoate (hereinafter, also referred to as “PHA” for short) that comprises a novel structural unit and a process for producing the same. More particularly, the present invention relates to a novel biodegradable PHA that comprises 3-hydroxyalkanoic acid units having a substituted or unsubstituted (phenylmethyl)sulfanyl group at the end of the side chain thereof, and to a process for producing PHAs from an alkanoic acid having a substituted or unsubstituted (phenylmethyl)sulfanyl group at the end of the side chain thereof by using a microorganism capable of producing PHA and accumulating it in the cell.
2. Related Background Art
It has been reported that various microorganisms can produce poly-3-hydroxybutyrate (hereinafter, also referred to as “PHB” for short) or other PHA and accumulate it in the cell (“Biodegradable Plastics Handbook”, Biodegradable Plastics Society Ed., NTS, pages 178-197 (1995)). These polymers may be utilized for production of various products by, for example, melt processing as with conventional plastics, but unlike many conventional synthetic polymer compounds, these polymers do not cause pollution in the natural environment because they are biodegradable, i.e., they are completely degraded by microorganisms in the natural world. Furthermore, they have good biocompatibility and their applications in the medical field as soft materials are expected.
Microbial PHAs are known to have different compositions and/or structures depending on, for example, the type of the microorganism, compositions of the culture medium, and culture conditions. Thus, studies have been done to control the composition and structure to improve physical properties of PHA.
(1) First, the following articles report or disclose synthesis of PHA by polymerization of relatively simple monomer units such as 3-hydroxybutyric acid (hereinafter, abbreviated as 3HB).
For instance, Alcaligenes eutrophus H16 (ATCC No. 17699) and mutants thereof are known to produce copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (hereinafter, abbreviated as 3HV) with various composition ratios (Japanese Patent Publication No. 6-15604 and Japanese Patent Publication Nos. 7-14352 and 8-19227.)
Japanese Patent No. 2642937 discloses production of PHA of C6 to C12 3-hydroxyalkanoate monomer units by feeding acyclic aliphatic hydrocarbon compounds as substrates to Pseudomonas oleovorans (ATCC No. 29347).
Japanese Patent Application Laid-Open No. 5-7492 discloses a process for producing a copolymer of 3HB and 3HV using a microorganism such as Methylobacterium sp., Paracoccus sp., Alcaligenes sp., and Pseudomonas sp. in contact with C3 to C7 primary alcohol.
Japanese Patent Application Laid-Open No. 5-93049 and Japanese Patent Application Laid-Open No. 7-265065 disclose production of two-component copolymers of 3HB and 3-hydroxyhexanate by cultivating Aeromonas caviae with oleic acid or olive oil as a substrate.
Japanese Patent Application Laid-Open No. 9-191893 discloses that Comamonans acidovorans IFO 13852 produces polyester containing 3HB and 4-hydroxybutyrate as the monomer units when it is cultivated in the presence of gluconic acid and 1,4-butanediol as substrates.
The above-mentioned PHAs are “usual PHAs” including monomer units having an alkyl group as the side chain thereof, synthesized by microorganisms via β-oxidation of hydrocarbons etc. or via fatty acid synthesis from saccharides.
(2) However, “unusual PHAs”, i.e., PHAs having a substituent other than an alkyl group on the side chain, are expected to be very useful when more extensive application of microbial PHAs is considered, for example, as functional polymers. Certain microorganisms have already been known to produce such “unusual PHAs”, and it has been tried to improve physical properties of microbial PHA with such an approach.
Examples of the substituents include unsaturated hydrocarbons, ester groups, cyano groups, halogenated hydrocarbons, epoxides, and those containing an aromatic ring or rings. Of these, PHAs having an aromatic ring have been studied actively.
For example, Makromol. Chem., 191, 1957-1965 (1990), Macromolecules, 24, 5256-5260 (1991), and Chirality, 3, 492-494 (1991) report that Pseudomonas oleovorans produces PHAs containing 3-hydroxy-5-phenylvalerate (hereinafter, abbreviated as 3HPV) as the monomer unit, where changes in physical properties of the PHA are observed probably due to the presence of 3HPV.
Of the PHAs having a substituent on the side chain thereof, lately those having a phenoxy group on the side chain have been actively developed.
It has been reported that Pseudomonas oleovorans produces from 11-phenoxyundecanoic acids PHA made with monomer units of 3-hydroxy-5-phenoxyvalerate and 3-hydroxy-9-phenoxynonanoate (Macromol. Chem. Phys., 195, 1665-1672 (1994)).
Macromolecules, 29, 3432-3435 (1996) reports production of PHA having monomer units of 3-hydroxy-4-phenoxybutyrate and 3-hydroxy-6-phenoxyhexanoate from 6-phenoxyhexanoic acids; production of PHA having units of 3-hydroxy-4-phenoxybutyrate, 3-hydroxy-6-phenoxyhexanoate, 3-hydroxy-4-phenoxybutyrate, 3-hydroxy-6-phenoxyhexanoate and 3-hydroxy-8-phenoxyoctanoate from 8-phenoxyoctanoic acid; and production of PHA made with units of 3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-7-phenoxyheptanoic acid from 11-hydroxyundecanoic acid, by using Pseudomonas oleovorans. 
Can. J. Microbiol., 41, 32-43 (1995) reports production of PHAs containing 3-hydroxy-6-(4-cyanophenoxy)hexanoic acids or 3-hydroxy-6-(4-nitrophenoxy)hexanoic acid as the monomer units by Pseudomonas oleovorans ATCC 29347 or Pseudomonas putida KT 2422 using octanoic acid and 6-(4-cyanophenoxy)hexanoic acid or 6-(p-nitrophenoxy)hexanoic acid as a substrate.
Of unusual PHAs developed, production of those having sulfur atoms in the form of sulfide (—S—) in the side chain thereof is reported in Macromolecules, 32, 8315-8318 (1999), where Pseudomonas putida 27N01 produced PHAs containing 3-hydroxy-5-(phenylsulfanyl)valeric acid and 3-hydroxy-7-(phenylsulfanyl)heptanoic acid as the monomer units, using octanoic acid and 11-(phenylsulfanyl)undecanoic acid as the substrates. In that case, the Pseudomonas putida 27N01 is pre-cultured in a culture medium containing octanoic acid only as the growth substrate, and then transferred to a culture medium that contains only 11-(phenylsulfanyl)undecanoic acid as a substrate.
Also Polymer Preprints, Japan Vol. 49, No. 5, 1034 (2000) reports production of PHAs containing two monomer units of 3-hydroxy-[(phenylmethyl)sulfanyl]valeric acid and 3-hydroxy-7-[(phenylmethyl)sulfanyl]heptanoic acid, by using Pseudomonas putida 27N01 and 11-[(phenylmethyl)sulfanyl]undecanoic acid as a substrate. In this case also, Pseudomonas putida 27N01 is precultured in a culture medium that contains only octanoic acid as the growth substrate, and then transferred to a culture medium that contains only 11-[(phenylmethyl)sulfanyl]undecanoic acid.
Concerning PHAs containing a 3-hydroxy-ω-[(phenylmethyl)sulfanyl]alkanoic acid unit among unusual PHAs, the above articles are the only reports on the biosynthesis of such PHAs. Further, the available production process is limited. Accordingly, the resulting polymers are not sufficient in types, purity, and yield. In the above process for producing the PHAs containing a 3-hydroxy-ω-[(phenylmethyl)sulfanyl]alkanoic acid unit, the polymer production is conducted by culturing the microorganism in a culture medium containing only ω-[(phenylmethyl)sulfanyl]alkanoic acid having a long carbon chain as the substrate, where ω-[(phenylmethyl)sulfanyl]alkanoic acid is also used as the growth substrate. Therefore, it is difficult to control the structure of the polymer.
PHAs containing a substituted 3-hydroxy-ω-[{[(substituted phenyl)methyl]sulfanyl}alkanoic acid unit that has a substituent such as various functional groups on the benzene ring of (phenylmethyl)sulfanyl group at the end of the side chain are PHAs having novel functionalities, and improvement in physical properties of such PHAs is predicted. Application of such PHAs will be expanded to novel fields where conventional PHAs have not been applicable. Thus, development of an efficient process for producing such PHAs is desired.